CXCL16/CXCR6 Chemokine Axis Mediates Progression Of Breast Cancer By PERK1/2-Dependent Mechanisms

Our previous studies demonstrate that CXCL6/CXCR6 chemokine axis induces prostate cancer progression by the AKT/mTOR signaling pathway. A number of studies indicate that CXCL16/CXCR6 chemokine axis is also involved in other tumor progressions, such as liver, ovarian, bladder cancer et al. However, its roles in breast cancer invasion and metastasis need further study. In the study, analysis of the CXCR6 expression in breast cancer and normal breast tissue microarray by immunohistochemical staining, the result shows the expression of CXCR6 is higher in breast cancer tissues than in normal breast tissues, expression of CXCR6 is significantly higher in metastatic lymph nodes than in normal lymph nodes, suggesting CXCR6 may be involved in the metastasis of breast cancer. Flow cytometric analysis of the expression of CXCR6 in a variety of breast cancer cell lines and the normal breast epithelial cell line HBL100, the result shows the expression of CXCR6 is higher in breast cancer cell lines than in HBL100 cells, and the highly metastatic MDA-231 cells shows the highest positive rate of CXCR6 expression. In order to reveal the role of CXCR6 in invasion and metastasis of breast cancer cells, CXCR6 stably overexpressing breast cancer cell lines were constructed by using of lentiviral system. Scratches healing and Transwell cell invasion assays show CXCR6 promotes migration and invasion of breast cancer cells. On mechanism researches, we found that CXCL16 / CXR6 chemokine axis does not affect the EMT process of breast cancer cells, but can significantly activate ERK1 /2 signaling pathway. When knockdown the expression of CXCR6 or treatment with U0126, the ERK1/2 pathway is inhibited and the cell invasion ability reduced, suggesting that CXCL16 /CXCR6 chemokine axis promotes migration of breast cancer cells by the ERK1/2 pathway dependent manner. The CXCR6-overexpressing and control cells gene transcriptional profiling analyses show the cytoskeleton related genes may play important roles in CXCR6 mediated process in breast cancer. Phalloidin staining assays showed that CXCR6 overexpression significantly increased F-actin formation, while CXCR6 knockdown significantly reduced F-actin formation. In addition, the formation of F-actin is regulated by the ERK1/2 and ROCK1/2 pathways. GST-pull down assays show that CXCL16/CXCR6 chemokine axis activates RhoA depending on the activation of the ERK1/2 pathway. Western blot analysis shows that CXCL16 /CXCR6 chemokine axis also inhibited the activity of actin depolymerization factor Cofilin, which may be mediated by RhoA. When MDA-231CXCR6 cells transfected withconstitutive activated mutant RhoAV14 plasmid or dominant negative mutant RhoAN19 plasmid, the formation of F-actin increased in RhoAV14 overexpressing cells while decreased in RhoAN19 overexpressing cells, suggesting the activity status of Rho A affects the stability of F-actin. Luciferase labeled breast cancer cells were injected into the tail veins of Nude BALB/c mice for 8 weeks, then the in vivo imaging system was used to monitor the metastasis of cancer cells; we found the number of lung micrometastases significantly reduced in CXCR6 knockdown mice. Immunohistochemical staining showed the ERK pathway was significantly activated in lung metastases sites of breast cancer. Taken together, our data shows for the first time that the CXCR6 / ERK1/2 / RhoA / Cofilin / F-actin pathway plays a central role in the development of BC. Targeting the signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for breast cancer.